PROJECT SUMMARY/ABSTRACT Formation of anti-citrullinated protein antibodies (ACPA) is an early step in the development of rheumatoid arthritis (RA). Serum ACPA are highly predictive of developing RA and can directly promote joint damage. Yet, there remains a major gap in our understanding of ACPA development, including where and how they are initially triggered. Importantly, to achieve the NIH's mission of disease/disability prevention in RA, an improved understanding of the initiating factors and pathways that lead to ACPA is needed as these factors/pathways would be novel targets for RA prevention. ACPA originate several years prior to the onset of arthritis in RA, and data support that they may initially develop in the lung mucosa. However, the mechanism by which the lung contributes to ACPA development is unclear. The overall hypothesis of the proposed project is that neutrophil extracellular traps (NETs) in the lung trigger ACPA and predict RA. This hypothesis is based on our published and preliminary data including our finding that NET remnants correlate with ACPA in the lung in subjects At- Risk for developing RA. Importantly, NET remnants are a composite of NET formation (NETosis) and NET clearance. This study will establish whether aberrancies in one or both of these processes contribute to ACPA generation in the lung and development of RA. In this project, induced sputum will be collected from subjects who are At-Risk for RA (based on known familial or serologic risk factors), healthy controls and subjects with RA. Using microscopy, the percentage of sputum neutrophils that undergo NETosis following cytokine stimulation will be calculated. The binding of serum ACPA to proteins on sputum NETs will also be measured. It is expected that subjects At-Risk for RA will have abnormally increased sputum NETosis following cytokine stimulation compared to controls. It is also expected that cytokine-induced sputum NETosis will correlate with sputum ACPA levels and that sputum NETs will express citrullinated proteins that bind serum ACPA from At- Risk subjects. The proposed project will also measure the degradation of NETs by DNase in sputum and the clearance of NETs by sputum macrophage phagocytosis. It is expected that both mechanisms of NET degradation and clearance will be decreased in subjects At-Risk for RA compared to controls and will correlate with sputum ACPA. In addition, At-Risk subjects will be followed for 3 years. Sputum ACPA and NET remnant levels will be measured at baseline and yearly to establish the ability of sputum NET remnants to predict which At-Risk subjects will develop RA. Mass spectrometry will also be used to identify citrullinated proteins present in sputum that are associated with development of RA. It is expected that increased sputum NET remnants mediated through sputum ACPA and unique cit-proteins will predict imminent RA in subjects At-Risk for RA. Ultimately, these findings can lead to novel approaches for RA prevention that target specific mechanisms of ACPA development (e.g. NET formation and clearance) at the site of initial immune dysregulation (e.g. the lung) in individuals who are At-Risk for RA.